The long-term goal of this proposal is to study the anatomical and pharmacological relationship of opioidergic system to GABAergic systems within the brainstem anti-nociceptive circuit of rats. Anti-nociceptive effects of opioids administered into ventral lateral periaqueductal grey matter, rostral ventral medulla and spinal cord dorsal horn appear to be mediate din part by neurons containing GABA. Our previous studies have demonstrated that neurons immunoreactive for GABA also express mu- opioid receptors in many brain regions, which suggests that opioids may directly affect GABA release. In addition, we have also reported that GAD-ir varicosities appose neurons expressing mu-opioid receptors, thus suggesting that opioid-sensitive neurons may be controlled by GABA. In preliminary studies, we have observed that GABA/A receptors in globus pallidus, hippocampus and DRG are co-localized with mu-opioid receptors. Thus, it appears that opioidergic and GABAergic systems in CNS are linked together. However, the anatomical and pharmacological relations between these two systems are poorly understood. Our studies will test the following hypotheses. 1). Co-expression of GABA-receptors and opioid receptors in neurons projecting from PAG to NRM, projecting from NRM to dorsal horn of the spinal cord, and neurons in the dorsal horn. This will be addressed by multi-color immunofluorescence using antibodies against one of the opioid receptors (mu-, addressed by multi-color immunofluorescence using antibodies against one of the opioid receptors (mu-, delta-, or kappa) combined with antibodies for GABA/A and GABA/B receptors. Immunocytochemistry will be combined with retrograde tract tracing technique. 2. Co-localization of GABA-receptors and endogenous opioid peptides and their relationship to neurons projecting from PAG to NRM, projecting from NRM to dorsal horn of the spinal cord, and neurons in the dorsal horn of the spinal cord. This will be addressed by employing multi-color immunofluorescence combining antibodies against GABA/A or GABA/B receptors with antibodies against Met-enkephalin, beta-endorphin, dynorphin/1-8 and endomorphin 2. 3. Pharmacological interactions between opioidergic and GABAergic systems. This will be done by employing radio-ligand binding assay in isolated membrane preparations of the ventral lateral PAG, the RVM (including NRM), and the dorsal portion of the spinal cord. Tissues will be dissected from the animals treated with either morphine (acute and chronic) or GABA. Results of this study will contribute to our understanding of the mechanisms of opioid anti-nociception, and will suggest strategies for developing alterative, non-opioid, approaches for pain-treatment.